The previously described Duchenne/Becker muscular dystrophy and cystic fibrosis are among the most common monogenic severe diseases. In the following, examples are shown for much more rare monogenic severe disorders, providing samples for the great variability for mutational spectrum, diverse clinical picture and methodological possibilities. The common feature of the diseases described is that, as the genes responsible for the diseases are cloned, prenatal molecular diagnostic procedures are applicable, especially, when the family-specific alteration is known (Factor V Leiden, being only a risk factor for venous thrombosis is excluded from this list).
1. Polycystic kidney and hepatic disease gene 1 (PKHD1).
Unlike the much more benign dominant counterpart, the autosomal recessive polycystic kidney disease (ARPKD) is a significant cause of neonatal morbidity and mortality accounting for a neonatal mortality rate of 25-35%.
Prevalence of the disease is 1:20,000 with a carrier frequency of 1:70. The gene that is responsible for the ARPKD encodes a large protein with unknown function (fibrocystin or polyductin), see in Figure 10.1.
Figure 10.1. Structure of the PKHD1 protein
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PKHD1 gene lacks any mutational hot spot, therefore, direct mutation analysis by gene sequencing is the recommended molecular genetic approach in the diagnosis of ARPKD.
2. Niemann Pick Type C disease (NPC).
The Niemann-Pick type C disease is rare monogenic disease that affects the intracellular transport of cholesterol. The disease affects quality of life to a great extent and is not curable. In 95% of the cases this disease is caused by mutations in the NPC1 gene. Figure 10.2. shows the predicted structure of the encoded NPC1 protein. The already known loss-of-function pathogenic mutations are labelled.
Figure 10.2. NPC1 protein
Figure 10.3. shows some amino acid residues affected by mutations casing Niemann-Pick type C disease. This domain is rich in cysteine amino acds, which are frequently involved in the formation of disulphide bridge.
Duble arrows show the already known pathogenic alterions. Amino acid reidues in red are phylogenetically conserved, which means that they cannot be replaced by other amino acids.
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Figure 10.3. Effects of the mutations: NPC1 gene.
3. Examples of inherited diseases in the blood coagulation system:
multifactorial and monogenic disorders
During the process of the humoral way of blood coagulation, a fibrin network is formed, which closes the wound. A key component of this process is the activation of the final effector enzyme, thrombin (T, Figure 10.4.).
Figure legends:
1. Activation of the procoagulant factor V (FV). Activation is done by thrombin or by active factor X (FXa). The non-enzymatic FVa is a cofactor of FXa in the prothrombin - thrombin conversion.
2. The thrombin-thrombomodulin complex activates the natural anticoagulant protein C (PC).
3. Factor V has anticoagulant properties too.
4. The FXa / FVa complex in the activated membrane surface activates prothrombin in the presence of Ca2+.
5. Activated protein C (APC) inactivates the procoagulant active factor V (FVa) in the presence of its cofactor, protein S by limited proteolysis that involves cleavage next to several arginine residues.
6. Procoagulant factor V can be inactivated by thrombin too.
7. Activated factor VIII is inactivated by APC/PS/FVac.
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Mutations in the gene encoding FVIII cause haemophilia A. Loss-of-function mutations in the protein C or protein S genes result in severe thrombotic disease, while different mutations in the gene for factor V might result in severe bleeding as well as thrombotic tendency. Inherited factor V deficiency, which is a result of loss-of-function mutation in the factor V gene show autosomal recessive inheritance. Prevalence of factor V deficiency is 1:1,000,000. Contrary to this, when a specific mutation affects the primary cleavage/inactivation site for APC, the arginine at position 506, will result in the opposite effect, that is, increased risk of thrombosis (thrombophilia). The consequence of this mutation (the so-called factor V Leiden mutation) is that the mutant active, procoagulant factor V will stay longer in the circulation leading to increased risk of thrombosis. Heterozygous genotype increases the risk by 5-10-fold compared to wild type individuals, while homozygous mutant genotype increases the risk of venous thrombosis by 50-100-fold.
The mutation being highly prevalent, it is also important at the level of public health. In Hungary, 1 out of 10 is heterozygous. Molecular testing of Factor V Leiden is one of the most commonly performed diagnostic assays in the developed world.
Figure 10.4. Blood coagulation: The protein C / protein S / Factor V system For the final proof of a given inherited disease, molecular genetic assays are frequently complemented with protein-based tests, as it has been
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demonstrated in the case of Duchenne/Becker muscular dystrophy. Such an analysis is shown in Figure 10.5. The figure shows the result of antigen measurement in the case of a family with a factor V deficient member (generation IV, arrow). As has been shown, the autosomal recessive factor V deficiency is a rare disease with a prevalence of 1:1,000,000. Two mutations are needed to develop the symptoms, inherited in trans. According to the situation shown in the picture, on the father’s side, the mutation can be traced back to the paternal grandmother (II. 2). Persons with heterozygous genotype will not have symptoms, as factor V levels around 50% are enough to maintain coagulation. The other disease-causing mutation (or more precisely, its consequence, the decreased factor V amount) can only be seen in the proband's mother (III. 2), which indicates its de novo generation.
Antigen levels show that factor V deficiency in this family is a result of the decreased amount of factor V protein (CRM-, cross reactive material - ), meaning that the mutations interfere with the expression or stability of the mutant factor V.
Figure 10.5. Complementer tests for molecular genetic analysis:
quantifcation of proteins by ELISA
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11. Pharmacogenetics
One of the most quickly developing field of molecular testing is pharmacogenetics. In the process of metabolism, drugs will be more water soluble, therefore more accessible for renal excretion. During drug metabolism, sometimes toxic compounds are formed. In many cases, metabolism is responsible for the activation of the prodrug. This process can be divided into two different types of reaction. Type I reactions are oxydation, reduction and hydrolysis. Type II (conjugation) reactions include sulfation, methylation, glucuronidation, acetylation. Both reactions – whose names do not indicate the succession of the reactions – normally make the originally lipophilic compound more hydrophilic (Figure 11.1.) The genes coding the proteins responsible for these processes are usually highly polymorphic. This means that in some cases, the individual response after the administration of a specific drug can be attributed to the genetic background of the patient. It also means that in some cases, the knowledge of the patient’s genetic status makes individualized therapy possible.
Individualized therapy has two major goals: it might help not only in quickly establishing the correct dose of certain drug, but also in avoiding the dangerous, sometimes life-threatening side effects.
Some pharmacogenetic examples are shown below.
Figure 11.1. Drug metabolism and excretion
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1. CYP2D6.
CYP2D6 is involved in the metabolism of many drugs, therefore it is one of the main pharmacogenetic targets (Figure 11.2.). Its gene is located in the chromosome 22. Null alleles (mutations that lead to non-functioning protein product or no protein product al all), labelled with white boxes will lead to the poor metabilzer (PM) phenotype. 5-10% of the Caucasian population belongs to this group. When a normal dose of the drug is administered to these patients, some severe side-effect might be experienced as a consequence.
There are mutations which decrease enzyme activity, even though they do not completely abolish it (dotted boxes). Patients (5-10% of the Caucasian population) with this genotype show intermediate (IM) phenotype. Side-effects are also expected, though to a lesser extent compared to the poor metabolizers. In the case of wild type alleles (black boxes) the resulting enzyme activity is normal. Those individuals (65-80% of the Caucasian population) are the extensive metabolizers (EM). The duplication and multiplication of the CYP2D6 gene might be present in 5-10% of the Caucasian population, resulting in ultrarapid metabolizer (UM) phenotype.
The administration of the normal dose of the drug is completely ineffective in those patients. The right-hand side of the picture shows the plasma concentration of the drug and its therapeutic range.
Figure 11.2. Genotype-phenotype associations in the case of CYP2D6
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2. Thiopurine methyltransferase (TPMT) polymorphisms.
Thiopurine methyltransferase (TPMT) together with xanthine oxidase are responsible for the degradation of the purine analogue drugs, including 6-thioguanine, 6-mercaptopurine and azathioprin. There are significant interindividual and ethnic differences between the enzyme activities. It is the polymorphism of the TPMT gene that is responsible for these differences (Figure 11.3.).The TPMT gene is located to chromosome 6 (6p22.3). It consists of 10 exons and 9 introns. Numerous different alleles are known, which differ from each other only in a few nucleotides. The most common (wild type) allele is TPMT*1. The presence of any other allele results in decreased enzyme activity, both in heterozygous, and in homozygous form.
The prevalence of heterozygosity is 11% among Caucasians. 1 in 300 is homozygous (phenotypically). The most common known alleles are TPMT*3A, 3B, 3C, 3D, 2, 4, 5, 6, 7, of which TPMT*3A-D are the most prevalent.
Individuals with either low or intermediate enzyme activity (homozygous or heterozygous genotypes, respectively) require much less than the standard dose of the above-mentioned drugs. The administration of the normal dose of the drug might result, especially in the homozygotes, inlife-threatening side effects, such as myelosuppression and pancytopenia. The different common TPMT alleles are the followings: TPMT*1 is the wild type.
TPMT*2 allele is a G238C replacement in the exon 5, resulting in an alanine-proline amino acid substitution. In TPMT*3A two point mutations are present in one allele: G460A in exon 7 (effect: alanine-threonine replacement) and A719G in exon 10 (tyrosine-cysteine substitution). G460A alone is TPMT*3B allele, while A719G is TPMT*3C. TPMT*3D allele has G292T mutation (exon 5, with a glutamine-stop consequence) in addition to the two mutations present in TPMT*3A. In TPMT*4 allele, the boundary of intron 9 - exon 10 is affected, with a G>A nucleotide substitution. TPMT*7 allele is T681G mutation in exon 10, resulting in a histidine-glutamine amino acid replacement.
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Figure 11.3. Human TPMT mutations
3. The pharmacogenetic aspects of the anticoagulant terapy.
The most commonly used oral anticoagulant worldwide is warfarin. Its administration is lifelong in some patients groups (e.g., for those with artificial heart valves). One of the sister drugs of warfarin is acenocoumarol (syncumar), which is very commonly used in Hungary (the use of warfarin is app. 10-15%). Warfarin interferes with the vitamin-K cycle (Figure 11.4.).
The Vitamin-K cycle is a critical process for the posttranslational modifications of some proteins (the majority of them are involved in blood coagulation). The vitamin-K cycle makes possible the generation of gamma-carboxy-glutamate residues in these proteins, which is necessary for the membrane binding. Such proteins are factor II (prothrombin), factor VII, factor IX, factor X, protein C and protein S. In the absence of correct gamma-carboxylation the membrane binding, and as a consequence the blood coagulation is severely impaired. One of the key enzymes of the vitamin-K cycle is VKOR (vitamin-K epoxide reductase). The main function of VKOR is the reduction of the epoxide form of vitamin-K. This is a two-step process, where first the kinon, then the hydrokinon is formed. Warfarin interferes with both steps, as it binds to the same site where vitamin-K does and this site cannot accommodate both at the same time. INR is used for monitoring the effect of warfarin.
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Pharmacogenetics of the warfarin/syncumar type of anticoagulant drugs is important from the point of view of both individual and public health. Both drugs exist in a mixture of optical isomers, but there are substantial differences. In the case of warfarin, the S isomer is responsible for most part of the anticoagulant effect, while in syncumar, it is the R. The key enzyme of the metabolism of the S isomers is CYP2C9. It has two common variants with decreased enzyme activity (CYP2C9*2 and *3 alleles). Although the VKORC1 gene is highly polymorphic, testing one functional variant, namely -1639G>A is sufficient. A wide international collaboration has resulted in a warfarin dosing algorithm, which was based on - in addition to other factors - the pharmacogenetic data. This dosing formula is available as an Excel worksheet.
In conclusion, the genetic status of CYP2C9 will affect the availability of the active drug (warfarin) and the genotype of VKORC1 will affect the efficiency of the vitamin-K cycle.
Figure 11.4. Pharmacogenetic aspects of vitamin-K cycle
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